Transformation of legumes
The Fabaceae (Leguminosae) family is the most important dicot plant family in the world. Because of its huge economic significance, much effort has been invested in improving agronomic traits by genetic engineering.
Agrobacterium-mediated transformation is a commonly employed method for transferring genes into plants. The plant species that until now have been successfully transformed by Agrobacterium are exclusively dicots (as opposed to monocots), but not all dicots are easily transformed.
Some plant families, for example Solanaceae, have been demonstrated to be particularly well suited for Agrobacterium-mediated gene transfer, while other families, such as the Fabaceae, are notorious for being recalcitrant.
Production of transgenic soybean plants (Glycine max) has been attempted in a variety of ways. Leaves and protoplasts have been used as explant sources, but no regeneration into transformed plants has been obtained in this manner. Cotyledons of soybean inoculated with Agrobacterium tumefaciens resulted in transgenic plants, but only one of the numerous tested genotypes was successfully transformed in this way (Hinchee et al., Bio/Technology 6:915, 1988). WO 94/02620 describes a method for producing transgenic soybean plants using hypocotyls or cotyledonary nodes and a series of steps specially designed for soybean transformation, including particular temperatures, pH values and Agrobacterium concentrations.
The use of cotyledons as explants is not, however, generally applicable to legume transformation, and other explant sources have been used in most cases. For example, for pea (Pisum sativum) transformation, explants from shoot cultures and seedling epicotyls have been employed as explants, and transgenic callus thus obtained was after 6 months regenerated to plants (Puonti-Kaerlas et al., Plant Cell Rep. 8:321, 1989).
For white clover (Trifolium repens) transformation shoot tips were inoculated with Agrobacteria, and transgenic plants were obtained (Voisey et al., Plant Cell Rep. 13:309, 1994).
Attempts have been made to transform a number of other legumes. For example, Phaseolus vulgaris cotyledonary nodes and hypocotyls incubated with Agrobacterium tumefaciens resulted in transgenic calli but no transgenic plants (McClean et al., Plant Cell, Tissue & Org. Cult. 24:131, 1991). Likewise with the genus Vigna (Garcia et al., Plant Science 48:49, 1986). No transgenic plants of peanut (Arachis hypogaea) have been reported despite considerable effort.
The present inventors have tried to transform guar using the soybean cotyledon procedure described by Hinchee et al., but were unsuccessful. Together with the results reported by other researchers, this demonstrates that the choice of transformation method for legumes is fully empirical, and that no general scientifically based guidelines can be deduced. Thus, the transformation procedure and explant source for transformation of a legume have to be developed according to the particular requirements of the genus, species, or even genotype, in question.
The numerous reported attempts to obtain transgenic plants of various legumes clearly show that transformation of legumes is very difficult, even to a scientist skilled in the art. This is further evidenced by the fact that of the approximately 100 legume species of commercial interest, less than 5 species have been transformed. Thus, successful transformation of a previously untransformed legume genus or species is anything other than routine.
Guar
Guar (Cyamopsis tetragonoloba) is a legume of significant commercial interest due to the high content of galactomannan in the seeds. Guar galactomannan is also known as guar gum and is used as an viscosity enhancer for both food and nonfood purposes.
The galactomannan is found in the endosperm, which makes up about 35% of the dry weight of the seed, 80-90% being pure galactomannan. Large endosperms are an unusual feature in Fabaceae, where the endosperm fraction of the seeds is predominantly absent or rudimentary; instead food reserves for germination in legumes are most often deposited in enlarged cotyledons.
None of the legume species with large galactomannan containing endosperms have been reported to have been genetically transformed.
Sulbactam
An inherent drawback of Agrobacterium-mediated gene transfer is the fact that the bacteria continue to grow after transformation. In order to prevent overgrowth of the plant material, the bacteria must be effectively eliminated, normally by addition of a penicillin-like antibiotic (.beta.-lactams) such as carbenicillin, cefotaxime, etc.
The penicillin-like substances are chosen because they are in principle non-toxic to plant tissues. In practice, however, these compounds often exert a considerable toxic effect on the explants. One possible reason for the phytotoxicity, aside from possible direct toxic effects, may be that the antibiotics are gradually degraded during the long incubation time in the presence of both bacteria and plant tissues. An example of an undesirable degradation product is from the very commonly used antibiotic carbenicillin, which can be degraded to phenylacetic acid. Phenylacetic acid possesses auxin-like properties and consequently gives increased callus growth on the explant, which in turn may impair regeneration.
Thus, it would be highly desirable to be able to use smaller amounts of antibiotics and/or antibiotics that do not have such undesired side effects. In the course of their work on the transformation of guar, the inventors found that the .beta.-lactamase inhibitor sulbactam dramatically reduces the required concentrations of penicillin-like substances, thereby improving transformation efficiency and reducing costs significantly.
This novel approach to control Agrobacteria growth is generally applicable to transformation of plants because it is related to the bacteria which must always be eliminated during transformation, and is thus not limited to any particular plant species.